Selective Chemokine Modulation

ABSTRACT

A body fluid purifying arrangement comprises a purifying chamber having a fluid input and a fluid output, a first filter arranged in connection the fluid input, and a second filter arranged in connection with the fluid output. In another embodiment, a body fluid purifying arrangement in the form of a syringe comprises a purifying chamber having a common fluid input and fluid output, and a filter arranged in connection with the common fluid input and output. Granules or particles made of a metal or an oxide of a metal are retained within the purifying chamber by the respective filter(s) to purify a body fluid passing through the purifying chamber from 10 kDa interferon-γ inducible protein, IP-10, wherein said metal is a metal of group 4 or 5 of the periodic table of the elements and selected from the group consisting of titanium, zirconium, hafnium, niobium and tantalum.

TECHNICAL FIELD

The present invention generally relates to selective modulation of thechemokine IP-10 and to treating or preventing diseases and pathogenicconditions characterized by adverse IP-10 expression.

BACKGROUND

Cytokines are a group of proteinaceous signaling compounds that are usedextensively for inter-cell communication. These compounds are criticalto the functioning of both innate and adaptive immune responses. Apartfrom their importance in the development and functioning of the immunesystem, cytokines play a major role in a variety of immunological,inflammatory and infectious diseases.

Cytokines are produced by wide variety of cell types (both haemopoieticand non-haemopoietic) and can have effects on both nearby cells orthroughout the organism, sometimes strongly dependent on the presence ofother chemicals and cytokines.

Each cytokine generally binds to a specific cell-surface receptor.Subsequent cascades of intracellular signaling then alter cellfunctions. This may include the upregulation and/or downregulation ofseveral genes and their transcription factors, in turn resulting in theproduction of other cytokines, an increase in the number of surfacereceptors for other molecules, or the suppression of their own effect byfeedback inhibition.

Chemokine refers to a specific class of cytokines that mediatechemoattraction (chemotaxis) between cells. These chemokines arepro-inflammatory activation-inducible cytokines that generally have amolecular mass of between 8 and 10 kDa. Their receptors are mainlyintegral membrane proteins containing seven membrane-spanning helices,which are coupled to G proteins.

Chemokines are released from a wide variety of cells in response tobacterial infection, viruses and agents that cause physical damage. Theyfunction mainly as chemoattractants for leukocytes, recruitingmonocytes, neutrophils and other effector cells from the blood to thesites of infection or damage. They can be released by many differentcell types and serve to guide cells involved in innate immunity and alsothe lymphocytes of the adaptive immune system. Some chemokines also haveroles in the development of lymphocytes, migration and angiogenesis.

As the cytokines and chemokines are involved in a multitude of differentdiseases and pathogenic or deleterious conditions, there is a generalneed of being able to effect or modulate the expression and/or releaseof these compounds. Furthermore, such release/expression modulationshould preferably be selective in terms of only affecting a limitednumber of target cytokines or chemokines.

Document [1] investigates the effect of titanium surfaces on macrophageactivation and secretion of proinflammatory cytokines and chemokines.When attached to rough titanium surfaces, lipopolysaccharide (LPS)stimulated macrophages increased their secretion of the cytokinesinterleukin-1β (IL-1β), IL-6 and tumor necrosis factor-α (TNF-α) and thechemokines monocyte chemoattractant protein-1 (MIP-1) and macrophageinflammatory protein-1α (MCP-1α).

Document [2] discloses that titanium particles stimulate the selectiveinduction of IL-8 and MCP-1 chemokines in human osteblast-likeosteosarcoma cells.

Document [3] investigates the effect of titanium particles on cytokinerelease by macrophage-like cells (MLC). Titanium particles significantlyenhanced MLC release of IL-1β, IL-8 and TNF-α.

Document [4] provides an overview of macrophage interactions withmodified material surfaces. The document discloses that macrophagescontacted with modified surfaces release of IL-1β, IL-6, IL-10 andTNF-α.

SUMMARY

The present invention overcomes these and other drawbacks of the priorart arrangements.

It is a general object of the present invention to provide a selectivemodulation of the chemokine 10 kDa interferon-γ inducible protein,IP-10.

It is another object of the invention to provide a composition that canbe used for purifying a sample from IP-10.

Yet another object of the invention is to provide a composition that canbe used for reducing IP-10 expression.

These and other objects are met by the invention as defined by theaccompanying patent claims.

Briefly, the present invention involves the use of a metal or a metaloxide having the capability of selectively binding IP-10 to its surface.The metal (oxide) further has an IP-10 downregulating effect in that acontact between the metal surface and an IP-10 producing cell will causea reduction in the IP-10 production of that cell.

The metal or metal oxide of the present invention is a metal of group 4or 5 of the periodic table of elements. Preferred such metals includetitanium, vanadium and tantalum and their oxides. More preferably, themetal is an oxide of titanium, such as titanium dioxide.

The metals and metal oxides of the present invention can be used formanufacturing a medicament for treating or preventing a diseasecharacterized by adverse expression and/or release of IP-10. Suchdiseases include adverse inflammatory responses, infectious diseases,autoimmune diseases, host versus graft diseases and foreign bodyreactions. In all these diseases, IP-10 is a key factor in thedevelopment of the diseases and a reduction of IP-10 can be used fortreating and/or preventing the diseases.

The method also involves methods of treating a disease characterized byadverse IP-10 expression and/or release in a subject. A first methodinvolves administering a medicament of the present invention to thesubject suffering from the disease. A second method involves an ex vivotreatment of a body fluid extracted from the subject. In this method,IP-10 is removed in an ex vivo filtering using the metals or metaloxides of the invention and the purified blood can then be returned tothe particular subject.

SHORT DESCRIPTION OF THE DRAWINGS

The invention together with further objects and advantages thereof, maybest be understood by making reference to the following descriptiontaken together with the accompanying drawings, in which:

FIG. 1 is a diagram illustrating the effect of grey titanium granules(GG) on the release of IP-10 in whole human blood;

FIG. 2 is a diagram illustrating the effect of pre-treatment (−1t)versus post-treatment (+1t) with grey titanium granules (GG) on therelease of IP-10 in whole human blood;

FIG. 3 is a diagram illustrating relative IP-10 gene expression in totalleukocytes and monocytes after incubating whole blood with titaniumgranules;

FIG. 4 is a diagram illustrating the effect of grey titanium granules(GG) on IP-10 gene expression in monocytes after incubating whole bloodwith titanium granules;

FIG. 5 is a diagram illustrating total and differential WBC count afterincubation with and without grey titanium granules (GG);

FIG. 6 is a diagram illustrating the concentration of IP-10 in a bloodloop system with untreated and infected (LPS) blood with or withouttitanium granules;

FIG. 7 is a diagram illustrating the IP-10 levels in a blood loop systemwith usage of grey titanium granules (GG), sifted gray titanium granules(GP), white titanium granules (WG) and ground white titanium granules(WP);

FIG. 8 is a diagram illustrating the IP-10 levels in a blood loopssystem using different amounts of added ground white titanium granules(WP);

FIG. 9 is a diagram illustrating the effect of ground white titaniumgranules (WP) on the IP-10 level in synovial fluid from patients withrheumatoid arthritis;

FIG. 10 is a diagram illustrating the effect of different amounts ofground white titanium granules (WP) on the IP-10 level in synovial fluid(SF) from a patient with rheumatoid arthritis;

FIG. 11 illustrates scanning electronic micrographs showing grey (A, C,E) and white (B, D, F) titanium granules (250×, 2000×, 5000×magnification);

FIG. 12 illustrates log differential mercury intrusion volume to poresize curves for grey (A) and white (B) titanium granules;

FIG. 13 is a diagram illustrating the effect of different titanium formsand other metals on IP-10 in serum spiked with IP-10;

FIG. 14 is a diagram illustrating the effect of different titanium formsand other metals on IP-10 in synovial fluid (SF);

FIG. 15 is a schematic illustration of a tool that can be used accordingto the present invention in scavenging IP-10 from a body fluid; and

FIG. 16 is a schematic illustration of another tool that can be usedaccording to the present invention in scavenging IP-10 from a bodyfluid.

DETAILED DESCRIPTION

Throughout the drawings, the same reference characters will be used forcorresponding or similar elements.

The present invention generally relates to a selective modulation ofspecific cytokines and chemokines. The present invention teaches thatcertain metals and metal oxides are able to reduce the expression andrelease of a particular chemokine and act as chemokine scavengers. Thiswas totally unexpected, as the prior art as illustrated by documents[1-4] all show that the metal titanium causes an upregulation in theproduction of cytokines and chemokines by different cell types.

The present invention can therefore be used for preventing and/ortreating diseased and medical conditions characterized by adverseexpression or release of the particular chemokine.

The relevant chemokine that can be reduced according to the presentinvention is 10 kDa (10 000 Dalton) interferon-γ inducible protein,IP-10. IP-10, also denoted C-X-C motif ligand 10 (CXCL10) or Crg-2 inthe art, belongs to the CDC chemokine family, which have a specificamino acid sequence of ELR (one letter amino acid code) immediatelybefore the first cysteine. These CXC chemokines induce migration ofneutrophils. However, IP-10 seems to differ from most CXC chemokines inthat it has no activity on neutrophiles and targets lymphocytesspecifically. IP-10 acts on the receptor CXCR3 as well as an IP-10specific receptor on epithelia and endothelia cells [5]. IP-10 issecreted by several cell types in response to interferon-γ (IFN-γ).These cell types include monocytes, endothelial cells and fibroblasts.

The metals and oxides of metals according to the present invention thathave the IP-10 reducing effect are metals and oxides of metals selectedfrom group 4 or 5 in the periodic table of the elements. Thus, thepresent invention encompasses the metals titanium (Ti), zirconium (Zr),hafnium (Hf), vanadium (V), niobium (Nb) and tantalum (Ta) and differentoxides of these metals. Preferred metals include titanium, tantalum andvanadium and their oxides, in particular titanium and oxides oftitanium.

Titanium has three oxidation state, Ti(II), Ti(III) and Ti(IV). Thepresent invention can use any of these oxides of titanium, i.e. Ti(II)oxide, Ti(III) oxide and Ti(IV) oxide. Ti(IV) oxide is also denotedtitanium dioxide (TiO₂) or titania in the art. This titanium dioxide isa preferred oxide form of titanium according to the present invention.TiO₂ can be present in different mineral or crystalline forms, includingrutile, anatase and brookite. Rutile is a tetragonal mineral usually ofprismatic habit, anatase or octahedrite is a tetragonal mineral ofdipyramidal habit, while brookite is an orthorhombic mineral. Apreferred titanium dioxide according to the present invention ispreferably in the rutile form or a mixture of the rutile and the anataseform.

A preferred oxide of zirconium is Zr(IV) oxide and Hf(IV) oxide is apreferred hafnium oxide. Vanadium is present in the oxidation statesV(II), V(III), V(IV) and V(V). Available vanadium oxides include V(IV)oxide (vanadium dioxide VO₂) and V(V) oxide (vanadium pentoxide V₂O₅).Niobium oxide can be in the form of Ni(V) oxide or Ni(III) oxide andtantalum has oxidation states of Ta(II), Ta(IV) and Ta(V).

An IP-10 reducing or removing metal agent according to the presentinvention comprises at least one metal of group 4 or 5 and/or at leastone oxide of a metal of group or 4. The metal agent could be a metal ormetal oxide in substantially pure form, such as at least about 95% metalor metal oxide, preferably at least 96, 97, 98 or 99% metal or metaloxide. The metal does not necessary has to be in pure form but can be indifferent chemical compounds or compositions, in addition to metaloxides, with the metal. Also alloys that comprise at least one metaland/or at least one metal oxide according to the present invention canbe used.

The metals and metal oxides of the present invention may reduce IP-10according to different fundamental mechanisms.

Firstly, the metals and metal oxides act like IP-10 scavenger in thatIP-10 in a surrounding medium will bind specifically to the surface ofthe metals and metal oxides. Thus, IP-10 will become enriched on themetal (oxide) surface and is therefore depleted from the surroundingmedium. This IP-10 metal (oxide) binding is highly specific as otherchemokines do not bind to the metals at the levels of IP-10, includingthe structurally and chemically closely related interleukin 8 (IL-8,also denoted CXCL8) that belong to the CXCL group of chemokines andmonocyte chemotactic protein 1 (MCP-1, also denoted CCL2) that has amolecular size in the range of IP-10 (the two chemokines contain 98 or99 amino acids). Blocking the metal (oxide) surface with BSA or fetalbovine serum does not affect the binding of IP-10 to the surface. Thebinding is also highly strong as it was not possible to remove boundIP-10 from the surface by added detergents (Tween-20).

The metal and metal oxides of the present invention act like highlyspecific IP-10 scavenger through the selective IP-10 metal (oxide)binding. As a consequence, the metals and metal oxides can be used forpurifying a medium from unwanted IP-10 and thereby removing or at leastreducing the concentration and amount of IP-10 in the medium.

Secondly, when contacting IP-10 producing cells with a metal or metaloxide of the present invention, the cell-metal interaction causes adownregulation in the IP-10 production as determined from reduced IP-10mRNA production. This in turn has the consequence of reducing the levelof newly produced IP-10. Experiments have also shown that the metals ofthe invention are indeed able to bind to known IP-10 producing cells,such as neutrophils and monocytes.

Thus, the metals and metal oxides of the present invention can reduceIP-10 by removing IP-10 from a medium through binding IP-10 and reducingthe amount of IP-10 expression by causing a downregulation of the IP-10mRNA production.

As the metals and metal oxides of the invention cause the IP-10reduction at least partly by binding IP-10 molecules to its metal(oxide) surface, the metals and metal oxides are preferably in a formthat has high specific surface area, i.e. surface area per weight unit.

The metal (oxide) agent can, in a first embodiment, be in the form ofporous granules, grains or granulates. The granules can be produced bythe well-known Hunter process or Kroll process. The resulting granulesare highly porous and have a large specific surface area. This specificsurface area of the porous metal granules is preferably at least 0.005m²/g, such as at least about 0.01 m²/g, more preferably about or morethan 0.02 m²/g, such as about 0.055 m²/g.

The preferred porosity of the granules implies that the granules includemultiple pores, including micro and/or macro pores, which are continuousthrough the granules, and openings of the multiple pores and ducts orpassages interconnecting at least a portion of the multiple pores. Theporosity of the metal (oxide) agent is preferably at least 25%, morepreferably at least 40%, such as at least about 50%. Highly porous metalgranules having a porosity of about or more than 70% can be manufacturedand used according to the present invention.

Instead of providing highly porous metal granules in mm or sub mm sizes,smaller metal (oxide) particles having an average diameter of 100 μm orless, such as a few μm or even smaller in the sub μm range, can be usedaccording to the present invention. Such small metal particles or dustwill provide a large surface area even at small quantities.

A first aspect of the present invention relates to the use of a metal ormetal oxide in the manufacture of a medicament for treating orpreventing a disease characterized by adverse expression and/or releaseof IP-10 in a subject. The metal is selected from at least one metal ofgroup 4 or 5 in the periodic table of elements, at least one oxide of ametal of group 4 or 5 or a mixture of at least one group 4 or 5 metaland at least one oxide of a metal of group 4 or 5.

The metal (oxide) agent of the present invention can both be used forpreventing diseases by being administered to a subject, preferablymammalian subject and more preferably a human subject, likely tosuffering a disease characterized by adverse IP-10 expression. In such acase, the metal agent will cause a reduction in the production of IP-10in the subject, by reducing the IP-10 mRNA expression. Furthermore,once/if IP-10 will be produced at high levels in the subject, thealready provided metal agent will bind IP-10 to its surface, therebyreducing the level of freely circulating IP-10 and preventing IP-10 fromhaving its deleterious effect of the disease. Thus, metal agent of theinvention will function as a safeguard that can prevent the onset of anIP-10 dependent disease.

Also a patient already suffering from an IP-10 dependent disease willbenefit from the medicament of the present invention as the metal agentwill remove already produced IP-10 through the metal-IP-10 binding. Inaddition, IP-10 producing cells will be stimulated to shut down or atleast reduce their IP-10 production.

The diseases or medical conditions that can be treated and/or preventedaccording to the present invention include IP-10 dependent diseasescharacterized by adverse (high) levels of IP-10 production in thesubject. The present invention is in particular useful for treating orpreventing adverse inflammatory reactions, where such a deleteriousinflammatory reaction can have vastly different causes, such asinfections, inflammatory diseases, foreign body reactions and hostversus graft diseases.

When a subject becomes infected, IP-10 is normally raised as a part ofthe immune defense against bacteria, viruses, parasites, fungi, prions,viroids, or other pathogens. However, at situations where the normalimmune system is altered, such as acquired immune deficiency syndrome(AIDS), the normal immune response can be devastating when the balanceis distorted. That is, the immune system is causing more damage to thetissue/body than the infection. In AIDS patients infected withchryptospordiosis, IP-10 was significantly increased and correlated tothe parasite burden. IP-10 was specifically localized to epithelialcells at the site of infection and when the infection was treated, IP-10levels were normalized. These results suggest IP-10 to be important forthe resolution of the infection in the normal immune defense, whereas inAIDS patients lacking effector cells, IP-10 may contribute to theimmunopathogenesis [6].

IP-10 may promote retroviral infection, such as human immunodeficiencyvirus (HIV), directly [7] or through recruitment of activated targetcells [8]. The level of IP-10 in the cerebrospinal fluid (CSF) isclosely associated with the HIV level in the CSF which suggests thatIP-10 is both a response to and a determinant of local infection [9].

The pathogenesis of the viral infection yellow fever (YF) is largelycontributed by cytokines and IP-10 was found to be significantly higherin fatal YF than in nonfatal YF. These results suggest cytokineintervention to be potential therapeutic strategies for treatment ofinfected patients [10].

Common cold is often induced by viral infection e.g. by rhinovirus.After rhinovirus infection of human epithelial cells it has been shownthat the epithelial cells produce IP-10 both in vitro and in vivo. Thelevel of IP-10 correlated to the severity of the symptoms and thereforeit is suggested that IP-10 play a role in the pathogenesis of viralinduced cold [11].

Thus, the present invention can be used for treating or preventingdifferent infectious diseases characterized by adverse IP-10 expression,in particular viral infectious diseases, including AIDS, HIV and yellowfever.

Autoimmune diseases are a particular form of inflammatory diseases,which results in an immune response against the body's own cells andtissues. IP-10 is a key player in immune responses, specifically indelayed type hypersensitivity (DTH) reactions. Such DTH reactionsinvolve autoimmune diseases. Therefore a reduction of IP-10 caused bythe metals and metal oxides of the invention can be an effective cure orpreventive measure for different autoimmune diseases. Examples ofautoimmune diseases that might be treated or prevented by the metalagent of the present invention include acute disseminatedencephalomyelitis (ADEM), Addison's disease, ankylosing spondylitis,antiphospholipid antibody syndrome (APS), aplastic anemia, autoimmunehepatitis, autoimmune oophoritis, Coeliac disease, Crohn's disease,diabetes mellitus type 1, gestational pemphigoid, Goodpasture'ssyndrome, Graves' disease, Guillain-Barré syndrome (GBS), Hashimoto'sdisease, idiopathic thrombocytopenic purpura, Kawasaki's disease, lupuserythematosus, multiple sclerosis (MS), myasthenia gravis, opsoclonusmyoclonus syndrome (OMS), optic neuritis, Ord's thyroiditis, pemphigus,pernicious anaemia, primary biliary cirrhosis, rheumatoid arthritis(RA), Reiter's syndrome, Sjögren's syndrome, Takayasu's arteritis,temporal arteritis (also known as giant cell arteritis), warm autoimmunehemolytic anemia and Wegener's granulomatosis.

MS is an autoimmune disease where the body produces antibodies againstthe myelin that protects nerves in the brain and spinal cord resultingin nerve loss. In patients with MS the spinal fluid contain high levelsof IP-10 and accumulation of T cells in the central nervous system isvery important in the pathogenesis of the disease. Therefore, IP-10 is apotential target in the search for MS therapies [12]. It has been shownthat IP-10 is concentrated to the lesions of the damaged nerve tissue[13-15]. Experiments have shown that the severity of the disease stateis correlated to the amount of expressed IP-10 [16], and blocking ofIP-10 (antibody treatment, DNA vaccine, antisense therapy andIP-10-bound immunotoxin) resulted in clinical improvement in differentmouse models [17, 18]. Usage of a metal agent according to the presentinvention can therefore be an effective medicament for treating orpreventing MS other known IP-10 blocking agents have had positiveeffects.

RA is an autoimmune disease where IP-10 has been shown to be active. Upto hundred-fold increase in IP-10 concentration has been detected insynovial fluid (SF) from RA patients [19]. This IP-10 may selectivelyattract T-cells to the SF and contribute to the pathogenesis of RA [20].IP-10 is induced in the SF by specific adhesion molecules andadministration of antibodies against these molecules significantlyinhibits IP-10 induction [21]. Data of IP-10, and other chemokines,receptor expression suggests that the chemokine system play a directrole in the destructive phase of RA [22]. A reduction in IP-10production and IP-10 levels caused by the present invention will bebeneficial in the treatment and prevention of RA.

In chronic hepatitis and autoimmune liver diseases, IP-10 is elevatedand reduced after successful IFN treatment. IP-10 plays a specific rolein the accumulation and death of hepatocytes in chronic hepaptitis [23].Similar results were obtained in patients with autoimmune liver disease[24].

IP-10 is up-regulated in skin lesions from patients with chronic discoidlupus erytematosus [25] and patients with systemic lupus erythematosus(SLE) have an increased IP-10 serum level and this IP-10 levelcorrelates to the level of disease activity [26]. Therefore, the metal(oxide) comprising medicament of the invention can be used also fortreating and/or preventing these types of autoimmune diseases.

The inflammatory response to be treated or prevented by the medicamentof the present invention can be due to an inflammatory disease of thegastro intestinal tract of a subject. Examples of such inflammatorydiseases include inflammatory bowel disease (IBD), ulcerous colitis (UC)and Crohn's Disease (CD, which is an autoimmune disease affecting thegastro intestinal tract). These diseases are severe chronic disorders ofthe gastro intestinal tract with unclear origin. Increasing evidencesuggest locally produced chemokines to play important roles in theprogression of the diseases [27]. IBD [28], UC [29] and CD [30] arecharacterized by an increase in IP-10 protein expression in the inflamedbowel and the therapeutic implication by inhibiting the IP-10 signalingpathway has been discussed [31]. Blockade of IP-10 signaling byadministration of antibodies against IP-10 resulted in protection fromacute colitis [32] as well as chronic colitis [33]. Butyrate inhibitsIP-10 release [34] and has been shown to be effective in the treatmentof UC patients [35]. Mice deficient of IFN-γ, where the IP-10 pathway isabolished, are unable to develop colitis in response to dextran sulphatestimulation which normal wild type mice does [28]. Antibodies againstIP-10 have been developed for the treatment of inflammatory boweldiseases [59]. These experimental results indicate that an IP-10inhibiting medicament of the present invention can be used for treatingand/or preventing inflammatory diseases of the gastro intestinal tract,including IBD, UC and CD.

The medicament of the present invention is also highly effective intreating and/or preventing inflammatory diseases. For example, IP-10 isup-regulated in skin lesions from patients with lichen (strong IP-10expression), chronic discoid lupus erytematosus (strong IP-10expression), allergic contact dermatitis (strong IP-10 expression), andpsoriasis (weak IP-10 expression) [25]. In these lesions a high amountof infiltrating active T cells was present suggesting a functionalinteraction between locally produced chemokines and CXCR3-expressing Tcells. Hence, the IP-10 pathway appears to play a significant role inthe recruitment and maintenance of T cell infiltrates in theinflammatory skin diseases. Further adverse inflammatory responses thatthe present invention may treat and/or prevent include glaucoma andinflammatory responses associated with glaucoma.

IP-10 has recently been shown to be produced and secreted by adipocytes[36] and has previous been found in atherosclerotic lesions togetherwith activated T cells [37]. Experimental studies on mice susceptiblefor atherosclerosis showed IP-10 to be a crucial key player in thelesion formation by local modulation of the immune system. These resultsin IP-10 deficient mice also showed that by inhibiting the IP-10 pathwaythere was a tendency to reduce the amount of pro-inflammatory T cellsand increase the protective T cell population which inhibits theatherosclerotic process [38]. These latest findings in animal modelssuggest that blocking chemokine/chemokine receptor interactions mayserve as a suitable approach to treat atherosclerosis. Likewise,chemokine antagonists that inhibit leukocyte recruitment couldparticularly be interesting to treat inflammation in response tomyocardial infarction, the major consequence of atherosclerosis [39].Therefore, the medicament of the present invention that results in areduced IP-10 expression and reduced IP-10 levels in a subject can beeffective for treating and/or preventing atherosclerosis.

Another adverse inflammatory response that is characterized by adverseIP-10 expression is asthma. In asthma mouse model IP-10 has been shownto contribute to the problematic hyper-reaction in the airways. IP-10deficient mice demonstrated the opposite results compared to wild typeanimals which indicates IP-10 pathway to be a target of asthma therapy[40, 41]. Chemokines and IP-10 also have an impact of other conditionsnegatively affecting the airways of a subject. Chronic obstructivepulmonary disease (COPD) is a condition which is characterized byirreversible airway obstruction due to narrowing of small airways anddestruction of the lung parenchyma. The condition causes airwayinflammation, involving neutrophil granulocytes, macrophages andlymphocytes. IP-10 is upregulated in the airways of COPD patients [42].The medicament of the present invention can therefore be used fortreating and/or preventing COPD and other inflammatory conditionsaffecting the airways.

When transplanting an implant into a subject body, a foreign bodyreaction will be triggered. This reaction is a special form of adverseinflammatory reaction caused by the introduction of foreign material,e.g. implant, into the subject. Such an inflammatory reaction ischaracterized by an increase in IP-10 secretion, and where IP-10 may bea key effector molecule in the inflammatory reaction. Thus, a reductionin IP-10 levels will reduce the inflammatory response to the foreignimplant and thereby inhibit the foreign body reaction.

The metals and metal oxides of the present invention can also be usedfor preventing or treating host versus graft diseases. Thus, theinvention can, be used in connection with graft transplantation forpreventing or at least reducing the risk of graft rejection, inparticular the acute phase of graft rejection. The outcome oftransplantation relies in principal on the reaction in the host to thegraft as rejection of the organ, tissue or cells is problematic totreat. In the acute rejection phase of a graft, IP-10 is increased andcan be used as a diagnostic marker of the rejection process since IP-10correlates to the severity of the rejection [43-46]. Experiments haveshown that transplantation of grafts in the presence of antibodiesdirected against IP-10 or grafts from IP-10 deficient mice resulted inlonger survival of the graft and fewer infiltrating T cells into thetransplant [47, 48]. The reverse has also been showed, i.e. graftssurvive longer in animals without ability to respond to IP-10 [49].Antagonists to the IP-10 signaling pathway have been developed andsuggested as a therapy that could improve the outcome oftransplantations [50]. Taken together these results indicate that theIP-10 scavenging and reducing metal agents of the present invention canbe very effective medicaments in treating or preventing graft rejectionof transplanted tissue grafts, organ grafts or cell grafts, such asislets of Langerhans.

Leukocyte infiltration is involved in several cancer types (neoplasia).These infiltrating leukocytes can be potential source of growth factorsfor the tumor cells and angiogenic factors for endothelial cells. IP-10has been shown to be a cytokine involved in such cancers. Furthermore,since chemokines are important mediators of leukocyte recruitment anddemonstrate altered characteristics of expression and activation inchronically inflamed tissue, they have been implicated as key regulatorsof inflammation and angiogenesis during cancer development.

Chronic activation of innate immune cells at sites of pre-malignanttumor growth may enhance tumor development. It has also become evidentthat early and persistent inflammatory responses observed in or aroundmany solid tumors, play important roles in establishing an environmentsuitable for neoplastic progression by providing diverse factors thatalter tissue homeostasis [51-53]

The teachings of the present invention can therefore be used fortreating or preventing cancer in a subject.

A second aspect of the present invention relates to a method of treatingor preventing a disease or disorder characterized by adverse expressionand/or release of IP-10 in a subject, preferably a mammalian subject andmore preferably a human subject. The method comprises administering ametal and/or an oxide of a metal to the subject suffering from thedisease, where the metal is a group 4 or 5 metal in the periodic tableof the elements.

In accordance with the invention, the metals or metal oxides can beprovided as pharmaceutically acceptable formulations using formulationmethods known to those of ordinary skill in the art. These formulationscan be administered by standard routes. In general, the copolymer may beadministered intravenously, intraperitoneally, subcutaneously, buccally,rectally, dermally, nasally, orally, tracheally, bronchially, topically,by any other parenteral route or via inhalation, in the form of apharmaceutical preparation comprising the active ingredient in apharmaceutically acceptable dosage form. The particular administrationroute to use will depend, among others, on the disorder or symptom totreat and can be determined by a physician. For example, a dermaladministration can be useful for treating inflammatory skin disorders,while an oral or rectal administration will be beneficial for subjectswith inflammatory reactions in the gastro intestinal tract.

In intravenous administration, the pharmaceutical medical compositioncomprises the metal or metal oxide of the invention in a solution of aselected solvent. In a particular administration implementation, themetal or metal oxide containing solution is injected once or preferablyat multiple time instants to a person in need of treatment. It couldalso be possible to employ a continuous or semi-continuous supply of themedicament from e.g. a medical pump or other administration equipment.Also administrations through so-called slow-release is possible andwithin the scope of the present invention.

In another particular implementation, a local administration in or inconnection with the inflammatory site can be used to allow a relativelyhigh local concentration of the active ingredient. This localadministration can be accompanied by one or more systemicadministrations.

In general, the formulations are prepared by uniformly and intimatelybringing into associate the active ingredient with preferably liquidcarriers or sometimes finely divided solid carriers or both, and then,if necessary, shaping the product.

Formulations suitable for parenteral administration include aqueous andnon-aqueous sterile injection solutions which may contain anti-oxidants,buffers, bacteriostats and solutes which render the formulation isotonicwith the blood of the intended recipient, and aqueous and non-aqueoussterile suspensions which may include suspending agents and thickeningagents. The formulations may be presented in unit-dose or multi-dosecontainers, for example, sealed ampoules and vials, and may be stored ina freeze-dried (lyophilized) conditions requiring only the addition ofthe sterile liquid carrier, for example, water for injections,immediately prior to use. The aqueous phase can be physiologic phosphatebuffered saline or other physiologic salt solution.

Formulations suitable for oral administration may be presented ascapsules, cachets or tablets each containing a predetermined amount ofthe active ingredient, as a powder or granules; as a solution or asuspension or emulsion in an aqueous liquid or a non-aqueous liquid.Formulations suitable for topical administration to the skin may bepresented as ointments, creams, gels and pastes comprising theingredient to be administered in a pharmaceutical acceptable carrier.Formulations for rectal administration may be presented as a suppositorywith a suitable base comprising, for example, cocoa butter or asalicylate. Formulations suitable for vaginal administration may bepresented as pessaries, tamports, creams, gels, pastes, foams or sprayformulations containing in addition to the active ingredient suchcarriers as are known in the art to be appropriate.

Examples of unit dosage formulations are those containing a daily doseor unit, daily sub-dose, as herein above recited, or an appropriatefraction thereof, of the administered ingredient.

The maximum allowable dosage that can be used according to the presentinvention depends, among others, on the particular disorder to treat,the particular patient, the severity of an inflammatory reaction and theadministration route. Experimental results have indicated that heattreated and ground titanium granules have an IP-10 binding capacity ofmore than 2000 pg IP-10/mg titanium particles for IP-10 spiked PBS,about 50 pg IP-10/mg titanium particles in IP-10 spiked serum and morethan 3.5 pg IP-10/mg titanium particles in synovial fluid. This numberscan be used by a physician in determining the amount of metal (oxide)particles to administer as the amount of IP-10 present in a body fluidis, for several medical disorders, proportional to the severity of thedisorder.

The metal agent of the invention is preferably provided (transplanted,injected or otherwise administered) in or in the neighborhood of thesite in a patient where a local IP-10 expression/release control ormodification is desired. This administration can be performed bytransplanting the granules or particles into or close to the targetsite. Alternatively, especially when utilizing granulates and powder,the metal (oxide) agent of the present invention can be injected intothe target site. As chemokines can be transported through the bloodsystem of a patient, the metal agent of the present invention can alsobe used for a general system delivery and still being able to affect theexpression and release of the selected target chemokine IP-10 from cellsin the blood system and vessels, in synovial fluid, cerebrospinal fluid,ascites or in lymph fluid.

An alternative method of treating or preventing a disease characterizedby adverse IP-10 expression/release involves an ex vivo treatment of abody fluid from a patient suffering from the disease. The method wouldinvolve extracting a body fluid from a subject suffering from thedisease. The body fluid contains adverse high levels of IP-10 and/orIP-10 producing cells. The body fluid could be blood, blood plasma,lymph fluid, cerebrospinal fluid, ascites and/or synovial fluid,depending on the particular treatment. The extracted body fluid iscontacted ex vivo with a metal or an oxide of a metal, where the metalis a group 4 or 5 metal. In this metal-fluid contact, IP-10 present inthe body fluid will bind to the surface of the metal (oxide) particlesand thereby be removed from the fluid. In addition, any IP-10 producingcells present in the extracted body fluid will, due to a cell-metalsurface contact shut down or at least reduce their IP-10 production. Thepurified body fluid can then be returned to the subject. The method issimilar to a traditional dialysis in that a body fluid is extracted andpurified, here from IP-10, before being returned to the body.

The fluid-metal contact can generally be very short as the binding ofIP-10 to the surface of the metal particles of the present invention.The contact time could be a few seconds up to some tens of minutes.

This form of treatment can be combined with other treatmentarrangements, in which a body fluid is extracted from a patient and thenreturned to the body. For example, a traditional renal or liver dialysiscan be combined with an extra purification step, in which IP-10 isremoved from the body fluid, typically blood. Also patients connected toheart and/or lung machines, such as extracorporeal membrane oxygenation(ECMO) machines can benefit from an additional IP-10 purifying stepaccording to the present invention.

This form of treatment is in particular advantageous for patientssuffering from a septic shock following an infection. A crucial step insaving such a patient is that the septic shock and the inflammatoryresponse that it causes must be combated as fast as possible. Suchpatients could be connected to a continuous ex vivo IP-10 dialysisaccording to the present invention. The level of IP-10 in the blood canthen be kept at a low level until further medications succeed inremoving the cause of the septic shock/infection.

The treatment method has several advantages as compared to administeringthe metal (oxide) agent to the subject body. Firstly, no metal (oxide)particles will be administered to the body and thereby any side effectsassociated with metal particle administration will not arise. Secondly,larger quantities of the metal particles and thereby a larger IP-10scavenging capacity can be utilized as compared to direct administeringto the subject body. Thirdly, the metal particles can be reused aftersuitable processing, in which bound IP-10 (and other molecules) havebeen removed from the particle surfaces.

FIGS. 15 and 16 schematically illustrate two possible arrangements ortools that can be used in connection with an ex vivo purifying of anextracted body fluid. In FIG. 15, the arrangement is basically in theform of a syringe 10 filled with metal (oxide) particles 1 of thepresent invention and being equipped with a filter 12 for preventing themetal particles 1 from leaving the syringe 10. During a treatment, abody fluid, such as synovial fluid, is drawn from a subject body intothe syringe 1. The fluid passes the filter 12 and comes into contactwith the metal particles 1. Any IP-10 molecules present in the fluidwill become attached to the particle surface and are thereby removedfrom the fluid. Following an incubation time, the fluid can be pushedback into the body (at the same or different site as the withdrawalsite). Due to careful choice of the filter pores, the particles 1 willremain in the syringe 10.

The filter pore size can be selected so that generally no cells presentin the extracted body fluid will pass through filter 12 and come intocontact with the metal particles 1. However, it can be advantageous ifIP-10 producing cells present in the body fluid are allowed to contactwith the metal particles 1 and thereby be subject to an IP-10 productionreduction due to the cell-particle contact. In such a case, the filterpore size is selected to allow such IP-10 producing cells, e.g.monocytes, endothelial cells and fibroblasts, but not the metalparticles 1 can pass through the filet 12.

FIG. 16 illustrates an arrangement 20 having a separate fluid input anda separate fluid output. The arrangement 20 forms a purifying chamberdefined by two filters 22 and 24. This purifying chamber is filled withmetal (oxide) particles 1 of the present invention. During operation,body fluid is pumped from a patient past the first filter 22. There thefluid comes into contact with the metal particles 1 and any IP-10present in the fluid will bind the particle surface. The purified fluidcan then, after incubation, be pumped past the second filter 24 and maythen be returned back to the body.

It is anticipated by the present invention that the filters 12, 22, 24employed in the arrangements of FIGS. 15 and 16 can be re-enforced byprotective layers that prevent the particles 1 from damaging the filters12, 22, 24.

The above described arrangements can also be employed for treatingextracted blood that is not intended to be returned to the same subject.Thus, the arrangements can be used for reducing the IP-10 level in, forinstance blood or plasma, handled by a blood bank. Such a method couldthen involve contacting a body sample, such as a body fluid, in vitrowith a group 4 or 5 metal or an oxide of a group 4 or 5 metal to therebyscavenging IP-10 from the body sample and preferably also reduce thelevel of IP-10 production in the body sample.

EXPERIMENTS Selective Cytokine and Chemokine Effect

Upon infection, the release of exogenous agents, e.g. LPS, and inductionof endogenous mediators, e.g. chemokines and cytokines, contributes tothe recruitment of circulating leukocytes to the inflamed tissue.Microbial products like LPS trigger multiple cell types to releasecytokines, which in turn are potent inducers of chemokines. Primarycytokines act as endogenous activators of the immune response, whileinducible chemokines act as secondary mediators to attract leukocytes[54]. Because of this complex interaction between cytokines andchemokines, it is important to evaluate not only the effect of metal(oxide) granules on the secretion of IP-10 but also on the release ofother chemokines and cytokines, which could give an indication ofsynergistic interaction among them in response to LPS and treatment withtitanium granules.

Incubation of Whole Human Blood Ex Vivo with Ti Granules

An ex vivo whole human blood model was used as previously described{55}. In brief, venous blood from healthy volunteers (n=7) wasanticoagulated with heparin (25 U/mL blood; Leo, Ballerup, Denmark) andthen incubated in microcentrifuge tubes at 37° C. with slow rotation inthe presence of lipopolysaccharide (LPS) (10 ng/mL blood; LPS wasderived from Escherichia coli serotype B6:026; Sigma, St. Louis, Mo.)and increasing dosages of grey titanium granules (0.015 g, 0.075 g,0.150 g, 0.300 g; Hereford Metal Powder Co Ltd, UK). Blood incubatedonly with LPS or saline was used as a positive and negative control,respectively.

Luminex Assay

At different time points (3, 6, and 24 h), plasma was obtained bycentrifugation at 7000 g for 3 min and was stored at −20° C. Plasmalevels of twenty-five different cytokines, see Table 1, were analyzedusing the solid phase sandwich multiplex bead immunoassays (Humancytokine 25-plex; Biosource International Inc., Camarillo, Calif., USA)according to the manufacturer's protocol. Briefly, primary antibodycoated beads and incubation buffer were pipetted into 96-well filterplates. The standards and samples were incubated in the presence of theprimary antibody beads for 2 h at room temperature on an orbital shaker.Following this, the wells were washed and biotinylated detectionantibodies were added. After further incubation for 1 h at roomtemperature, the wells were washed and streptavidin-phycoerythrinsolution was added to each well and incubated for 30 min at roomtemperature. Finally, the wells were washed thoroughly, sheath fluid wasadded and read using the Luminex xMAP system (Luminex Corporation,Austin, Tex., USA).

TABLE 1 measured cytokines IL-1β IL-1Ra IL-2 IL-2R IL-4 IL-5 IL-6 IL-7IL-8 IL-10 IL-12 IL-13 IL-15 IL-17 TNF-α INF-α INF-γ GM-CSF MIP-1αMIP-1β IP-10 MIG Eotaxin RANTES MCP-1 IL—interleukin INF—interferonIP—interferon inducible protein MCP—monocyte chemotactic proteinMIG—monokine induced by INF-γ MIP—macrophage inflammatory proteinTNF—tumor necrosis factor GM-CSF—granulocyte macrophage colonystimulating factor RANTES—regulated upon activation, normal Tcell-expressed and secreted

The striking result of the experiments was that the presence of thetitanium granules had a profound effect by almost completely shuttingdown the expression of IP-10 in a dosage dependant manner.

The titanium granules had no or merely minor effects on the othercytokines whose expression were induced by LPS. Interestingly, thesecretion of other chemokines from the same family (CXC chemokines) asIP-10, like IL-8, was not affected in the same way to IP-10 aftertreatment with titanium granules. Furthermore, chemokines having similarsize as IP-10, such as MCP-1, were not affected.

The negative control (saline) and the positive control (LPS only) showedthe predicted effects with normal cytokine levels in all samplesincubated with saline only, and dramatically increased cytokine levelsin samples incubated with LPS, mimicking the onset of an acuteinfectious inflammatory response. In samples incubated with titaniumgranules in the absence of LPS the cytokine levels were well within therange observed for the negative control, indicating that the titaniumgranules themselves did not elicit an inflammatory response.

Effect of Ti Granules on IP-10 Secretion in Whole Human Blood

The ex vivo whole human blood model previously described above and in[55] was used. Briefly, fresh venous blood of healthy volunteers (n=7)was added to different amounts of grey titanium granules (GG) and spikedwith LPS (10 ng/ml). After 3, 6 and 24 h, plasma was isolated andanalysed for IP-10 by Luminex assay. In other set of experiments, theblood was given either in pre- or post-treatment, by administeringtitanium granules 1 h before LPS, at the same time as LPS, or 1 h afterLPS.

As shown in FIG. 1, grey titanium granules reduced the production ofIP-10 in whole human blood of healthy volunteers, stimulated by LPS, ina dose-dependent manner. The results are significant in all the dosesand time points tested. Mean values±SEM of 7 donors are shown, *indicates significant differences versus LPS alone (p<0.05).

In order to find out whether grey titanium granules were more effectiveas pre- or post-treatment, titanium granules were given 1 h prior tostimulation with LPS (−1t), at the same time as LPS (0t), or 1 h afterstimulation with LPS (+1t) (n=3). Six hours after the addition of LPS,plasma was obtained and IP-10 levels were analysed by Luminex assay.

FIG. 2 shows that titanium granules were equally effective reducingIP-10 in blood that was pre-treated, or treated at the same time, andeven after LPS. Basal IP-10 levels (without LPS) were as wellsignificantly reduced in blood after the different incubation times withtitanium granules (GG), to the same extent as LPS-treated samples withtitanium granules (GG+LPS). Mean values±SEM of 3 donors are shown, *indicates significant differences of GG versus basal or LPS alone,respectively (p<0.05).

Effect of Ti Granules on IP-10 Gene Expression after Incubation withHuman Blood

The effect of Ti granules on IP-10 secretion was disclosed in theabove-presented experiments. It was then investigated whether titaniumgranules had any effect on IP-10 gene expression.

RNA Isolation

Total RNA was isolated from total leukocytes or monocytes afterincubating whole blood with titanium granules for 2 h, using RNeasy minikit (Qiagen, Valencia, Calif., USA), according to the manufacturer'sprotocol. In order to isolate monocytes, a pre-processing step beforethe cell lysis was included to separate monocytes from whole blood,using Dynabeads CD14 coated with anti-CD 14 monoclonal antibody(Invitrogen/Dynal, Carlsbad, Calif., USA). Total RNA was quantified at260 nm using a Nanodrop spectrophotometer (NanoDrop Technologies,Wilmington, Del., USA).

Real-time RT-PCR Analysis

Total RNA (0.5 μg) isolated from total leukocytes or monocytes wasreverse transcribed to cDNA at 42° C. for 60 min using iScript cDNASynthesis kit (BioRad, Hercules, Calif., USA) that contains botholigo(dT) and random hexamers. Each cDNA was frozen (−20° C.) inaliquots until the PCR reactions were carried out.

Real-time PCR was performed in the iCycler (BioRad, Hercules, Calif.,USA) using SYBR green detection. Real-time PCR was done for threehousekeeping genes: 18S ribosomal RNA (18S rRNA),glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and β-actin, and fourtarget genes: IP-10, IL-6, IL-10 and TNF-α. Table 2 lists the primersused and the parameters of the real-time PCT.

TABLE 2 Primers and PCR parameters SEQ ID T_(m) amplicon Amplicon GenePrimer sequence NO: (° C.) size (bp) IP-10S 5′-GCTACAATGAAAAAGAAGGGTGA-3′ 1 84.5 185 A 5′-TAGGGAAGTGATGGGAGAGG-3′2 IL-6 S 5′-AGGAGACTTGCCTGGTGAAA-3′ 3 84.0 196A 5′-GCATTTGTGGTTGGGTCAG-3′ 4 IL-10 S 5′-TTATCTTGTCTCTGGGCTTGG-3′ 5 84.0139 A 5′-ATGAAGTGGITGGGGAATGA-3′ 6 TNF-α S 5′-CTATCTGGGAGGGGTCTTCC-3′ 788.0 181 A 5′-GGGGGTAATAAAGGGATTGG-3′ 8 β-ActinS 5′-CTGGAACGGTGAAGGTGACA-3′ 9 85.5 136 A 5′-AAGGGACTTCCTGTAACAATGCA-3′10 18S rRNA S 5′-GTAACCCGTTGAACCCCATT-3′ 11 86.0 151A 5′-CCATCCAATCGGTAGTAGCG-3′ 12 GAPDH S 5′-TGCACCACCAACTGCTTAGC-3′ 1385.0 87 A 5′-GGCATGGACTGTGGTCATGAG-3′ 14 S Oligonucleotide sequence ofsense primer A Oligonucleotide sequence of antisense primer

Each reaction contained 5 μl of cDNA, 0.5 μM of the sense and antisensespecific primers, 12.5 μl of 2×IQ™ SYBR® Green Supermix (BioRad,Hercules, Calif., USA) in a final volume of 25 μl. The amplificationprogram consisted of a preincubation step for denaturation of thetemplate cDNA (3 min, 95° C.), followed by 40 cycles consisting of adenaturation step (15 s, 95° C.), an annealing step (15 s, 60° C.) andan extension step (30 s, 72° C.). After each cycle, fluorescence wasmeasured at 72° C. A negative control without cDNA template was run ineach assay. Samples were run in duplicate.

Real-time efficiencies were calculated from the given slopes in theiCycler software using serial dilutions, showing all the investigatedtranscripts high real-time PCR efficiency rates, and high linearity(r>0.99) when different concentrations were used. PCR products weresubjected to a melting curve analysis on the iCycler and subsequently 2%agarose/TAE gel electrophoresis to confirm amplification specificity, Tmand amplicon size, respectively.

In order to allow relative quantification after PCR, standard curveswere constructed from the standard reactions for each target andhousekeeping genes by plotting Ct values (cycle threshold), i.e. thecycle number at which the fluorescence signal exceeds background, versuslog cDNA dilution. The Ct readings for each of the unknown samples werethen used to calculate the amount of either the target or housekeepingrelative to the standard. Relative mRNA levels were calculated as theratio of relative concentration for the target genes relative to thatfor the mean between the three housekeeping genes (18S rRNA, GAPDH andβ-actin), to correct for RNA. Values were expressed as a percentage ofnegative control samples (saline), which were set to 100.

IP-10 gene expression was monitored in a) total leukocytes that remainedin suspension after incubation with titanium granules, b) totalleukocytes that attached to titanium granules after the incubation andc) monocytes isolated with beads coated with anti-CD 14 monoclonalantibody.

FIG. 3 illustrates the relative IP-10 gene expression in totalleukocytes and monocytes after incubating whole blood with titaniumgranules for 2 h. It is seen in FIG. 3, that the monocytes had thehighest IP-10 gene expression. Leukocytes that had attached to thegranules showed higher IP-10 gene expression (12-fold) than leukocytesthat remained in suspension.

Human monocytes were isolated for monitoring IP-10 gene expression withtitanium granules. As seen in FIG. 4, IP-10 mRNA levels weredown-regulated in monocytes after incubation with titanium granules (2 hincubation) and stimulated with LPS in the two donors tested.

Total and Differential White Blood Cell Count

Experiments were conducted in order to find out which types ofleukocytes that have the ability to attach to the surface of titaniumgranules after incubating with whole human blood.

After incubating blood with grey titanium granules for 2 h, as describedabove, total and differential white blood cell (WBC) counts wereperformed immediately in a hematology analyzer (Cell-Dyn 4000, AbbottDiagnostics Division, Santa Clara, Calif., USA).

FIG. 5 illustrates the total and differential WBC count (i.e.neutrophils, lymphocytes, monocytes, eosinophils and basophils,respectively) after 2 h incubation with and without grey titaniumgranules (GG). Titanium granules showed ability to bind neutrophils andmonocytes, which are known to be IP-10 producers.

Table 3 below lists the normal range for WBC count and the differentialwhite blood cells.

TABLE 3 Normal range of WBC count and differential WBC Number of cellsper L Percentage of total WBC WBC count 4.5-11 × 10⁹  Polymorphonuclear1.8-7.8 × 10⁹   50-70% neutrophils Band neutrophils 0-0.7 × 10⁹  0-10%Basophils 0-0.2 × 10⁹ 0-2% Eosinophils 0-045 × 10⁹  0-6% Lymfocytes1-4.8 × 10⁹ 15-45% Monocytes 0-0.8 × 10⁹  0-10%

Statistics

All data are presented as mean values±SEM. Differences between groupswere assessed by Student's t-test using the program SPSS® for Windows,version 14.0. Results were considered statistically significant at theP<0.05 level.

IP-10 Binding Capacity of Ti Materials

Recombinant human IP-10 was purchased from R&D Systems and IP-10 wasanalyzed using sandwich ELISA technique according to the manufacturer(R&D Systems). The effect of different titanium forms on chemokine levelwas studied in three different systems: a blood loop model, IP-10 spikedserum/PBS and in synovial fluid. Table 4 lists the titanium formstested.

TABLE 4 Investigated Ti forms Abbre- Particle Metal viation Compositionsize Manufacturer Ti granules GG 99.97%, >80% Ø~1 mm Hereford Metalsponge form Powder Co Ltd Heat treated WG 99.97% Ø~1 mm Hereford MetalTi granules Powder Co Ltd (900° C., 3 h) Sifted GG GP 99.97%, >80%<0.075 mm Hereford Metal sponge form Powder Co Ltd Ground WG WP 99.97% *Hereford Metal Powder Co Ltd * The resulting Ti particles generally hada size in the range of sub- micrometers up to 100 μm but with an averagediameter size of about 10 μm.

Blood Loop System

IP-10 was studied in a blood loop system (described previously [56,57]), with the exception that fully heparinized (20 U/ml, Leo Pharma)blood were used. Briefly, fresh human blood from healthy volunteers wascollected in surface-heparinized 60-ml syringes with a cannula(18-guage, Microlance; Becton Dickinson) that was connected to asurface-heparinized silicon tubing. During sampling, the syringe wasrotated continuously. Blood (7-8 ml) was then added to each loop (PVCtube, diameter 6.3 mm, length 39 cm) leaving an air volume of ˜4 ml.After the tubes were filled, the loops were closed with a heparinizedstainless-steel connector and placed on a rocking device at 37° C. Theloops were rocked at an amplitude setting that prevented the blood frommaking contact with the connectors. To initiate IP-10 production LPSfrom Escherichia coli (10 ng/ml, Sigma) was added. Titanium in differentamounts and different forms were added to the loops to investigate theeffect on IP-10 levels. After 10, 30, 60, 120 and 180 minutes sampleswere collected and plasma was stored at −20° C. until IP-10 analysis.

Spiked Serum/PBS

Serum was prepared from donated blood according to standard laboratoryprotocol. PBS was supplemented with 1% Bovine Serum Albumin (BSA) toprevent protein attachment to the plastic ware. Recombinant Human IP-10was added to the serum/PBS (200-2000 pg/ml) and samples (500 μl) wereincubated with different metals/oxides (20-200 mg) for 3 hours at roomtemperature on an orbital shaker (˜400 rpm). After incubation sampleswere centrifuged briefly (2 minutes at 10,000 g) and supernatants wereanalyzed immediately for IP-10 content or stored frozen (−20° C.) untilanalysis.

Synovial Fluid

Synovial fluid (SF) was obtained from patients with rheumatoid arthritisat the Karolinska Hospital (Solna, Sweden) or from Oslo Rikshospital(Norway). For 3 hours 150 μl SF was incubated in room temperature on anorbital shaker (˜400 rpm) with or without (10-40 mg/sample) groundedwhite granules (WP). When the effect of different metals/oxides wasstudied, 500 μl sample and 20 mg metal/oxides were used. Afterincubation, samples were centrifuged briefly (2 minutes at 10,000 g) andsupernatants were analyzed immediately for IP-10 content or storedfrozen (−20° C.) until analysis.

Blood infection was simulated by LPS addition to blood loops whichresulted in an increase in IP-10 production. After 2 hours the IP-10level in the LPS treated blood was raised and further elevated after 3hours. By adding untreated sponge titanium granules (GG) the IP-10levels were reduced in the infected blood, see FIG. 6. There was adose-response like reaction when different amounts of GG were added.Normal IP-10 levels were observed in uninfected blood after only GGaddition. The two negative controls, PBS or PBS with 200 mg titaniumgranules, showed no increase in IP-10 levels.

The effect of different forms of titanium granules on IP-10 levels wereanalyzed in the blood loop system. In whole blood both grey (GG) andwhite (WG) granules were found to be equally efficient in IP-10reduction. The powder forms of the granules, GP and WP, were moreefficient than whole granules and white powder (WP) had the highestIP-10 reducing capacity as is illustrated in FIG. 7. This figureillustrates the IP-10 levels in blood 180 minutes after LPS addition.The addition of 200 mg of white powder (WP) to 7 ml of infected bloodresulted in a totally abolished IP-10 response. PBS treated blood wasused as negative control.

In order to evaluate the IP-10 reducing capacity of grounded whitetitanium granules different amount of white powder (WP) were added toinfected (LPS-treated) blood and the IP-10 levels were analyzed 180minutes after the addition. IP-10 was found to be regulated in blood ina dose-response like fashion when WP was added, which is illustrated inFIG. 8. The addition of 66 mg of white powder to 7 ml of infected bloodresulted in a normalized IP-10 level.

The temporal binding of IP-10 to white powder in PBS solution wasstudied and already after 10 minutes could WP reduce IP-10 levels tozero (3 pg IP-10/mg WP). The reduction was non-reversible and sustainedfor as long as studied (24 hours). It was not possible to block thisbinding with 5% BSA or 10% fetal bovine serum and it was not reducedafter detergent addition (0.05% Tween-20).

The synovial fluid from inflamed joints in rheumatoid arthritis patientshas previously been shown to contain high levels of IP-10, which mayinfluence the pathogenesis of the disease [20, 58]. The IP-10 level insynovial fluid from five different patients was strongly reduced afteraddition of white granule powder (WP), see FIG. 9. The synovial fluidwas incubated with either 10 or 40 mg of WP for 3 hours at roomtemperature.

The IP-10 binding capacity of grounded white granules in synovial fluidwas studied in a system where increasing amounts of WP was added to theSF, see FIG. 10. An apparent dose-response relationship was observed inthese studies.

The capacity of IP-10 reduction of grounded white granules (WP) wascalculated for the different systems studied here. In spiked PBS >2000pg IP-10/mg WP was retained, in spiked serum about 50 pg IP-10/mg WP wasretained and in synovial fluid >3.5 pg IP-10/mg WP was retained.

Physical Characterization of Ti Granules Scanning Electron Microscope

A Scanning Electron Microscope (SEM, Philips XL 30 ESEM, FEI ElectronOptics, Eindhoven, Netherlands) was used to examine the surface of grey(GG) and white (WG) titanium granules.

Mercury Intrusion Porosimetry

The pore size distribution measurements of the grey (GG) and white (WG)titanium granules were performed using mercury porosimetry (Autopore IV9500, Micromeritics, Norcross, Ga., USA). The contact angle used was130°. The samples were evacuated for 10 min at an evacuation pressure ofabout 50 μmHg. The mercury filling pressure was about 0.22 psia.

FIG. 11 shows the surface of untreated titanium granules (grey granules,GG), see A, C and E, and heat-treated (900° C., 3 h) titanium granules(white granules, WG), see B, D and E, at magnifications of 250×, 2000×and 5000×. It is seen from the micrographs that heat treatment reducedthe number of pores in white granules. This is in accordance with theresults obtained with the total porosity, which was 73% and 57% for greyand white titanium granules, respectively. The total intrusion volume,total surface area, median pore diameter, bulk density, apparent densityand percent porosity for grey and white titanium granules are given inTable 5.

TABLE 5 intrusion data summary for grey and white Ti granules Grey TiWhite Ti Property granules granules Total intrusion volume (mL/g) 0.73940.4895 Total surface area (m²/g) 0.055 0.021 Median pore diameter(Volume) (μm) 180.0 328.7 Median pore diameter (Area) (μm) 21.0 28.9Average pore diameter (4 V/A) (μm) 53.7 93.3 Bulk density at 0.22 psia(g/mL) 0.9913 1.1614 Apparent (skeletal) density (g/mL) 3.7126 2.6916Porosity (%) 73.3 56.9

FIG. 12 illustrates log differential mercury intrusion volume to poresize for grey (A) and white (B) Ti granules. As shown in the figure,micropores (˜40 μm) present in grey granules were absent in whitegranules, while macropores (˜400 μm) were not affected by the heattreatment.

IP-10 Binding Capacity of Ti and Other Metals Materials

In order to investigate the IP-10 binding capacity of different titaniumoxides and other metals, two different systems: IP-10 spiked serum andin synovial fluid were used. Table 6 lists the other metals and oxidesthat were studies in addition to the titanium forms listed in Table 6.

TABLE 6 Investigated metals Composi- Particle Manufac- MetalAbbreviation tion size turer TiO₂ (80% Anatase/ TiO₂(80A/20R) 99.5% ~21nm Degussa 20% Rutile) Ti(IV) oxide Ti(IV)Ox(R) 99.9% <5 μm Aldrich(Rutile) TiO₂ (Anatase) TiO₂(A) — — Sachtleben Titaniumcarbide TiCarb —<2 μm Roth Ti(II) oxide Ti(II)Ox 99.9% <45 μm Aldrich Ti powder Ti ~32599.9% <45 μm Aldrich Ta powder Ta ~325 99.9% <45 μm Aldrich V powder V~325 99.5% <45 μm Aldrich Mn(II, III) oxide Mn(II, III)Ox   97% —Aldrich Ni(II) oxide Ni(II)Ox 76-77%  <10 μm Aldrich Cu(II) oxideCu(II)Ox   97% <5 μm Aldrich Fe(III) oxide Fe(III)Ox   98% <5 μm AldrichZn oxide ZnOx   99% — Fluka

20 mg of the different metals were added to serum spiked (200 pg/ml)with IP-10. FIG. 13 shows the results from these experiments. Among thedifferent titanium forms it can be concluded that white titanium (WP,Ti(IV)Ox(R), TiO₂(80A/20R), TiO₂(A)) is the most efficient form to lowerIP-10 levels in blood. Both tantalum and vanadium, which are closelyrelated to titanium, were efficient in IP-10 lowering. Neither zinc,copper nor manganese was affecting IP-10 levels in spiked serum to anyextent.

The effect of these different oxides and metals on IP-10 levels was alsoinvestigated in synovial fluid, see FIG. 14. These results did ingeneral confirm the results from IP-10 spiked serum with someexceptions. In SF Ti(II)oxid (black) and zinc were more efficient thanobserved in studies on serum, whereas the other metals affected theIP-10 levels to the same extent.

It will be understood by a person skilled in the art that variousmodifications and changes may be made to the present invention withoutdeparture from the scope thereof, which is defined by the appendedclaims.

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1. A body fluid purifying arrangement comprising: a purifying chamberhaving a fluid input and a fluid output; a first filter arranged inconnection said fluid input; a second filter arranged in connection withsaid fluid output; and granules or particles made of a metal or an oxideof a metal retained within said purifying chamber by said first filterand said second filter to purify a body fluid passing through saidpurifying chamber from 10 kDa interferon-γ inducible protein, IP-10,wherein said metal is a metal of group 4 or 5 of the periodic table ofthe elements and selected from the group consisting of titanium,zirconium, hafnium, niobium and tantalum.
 2. The body fluid purifyingarrangement according to claim 1, wherein said metal is selected from agroup of titanium and tantalum.
 3. The body fluid purifying arrangementaccording to claim 1, wherein said oxide of said metal is selected froma group consisting of an oxide of titanium and an oxide of tantalum. 4.The body fluid purifying arrangement according to claim 3, wherein saidoxide of titanium is a titanium dioxide.
 5. The body fluid purifyingarrangement according to claim 4, wherein said titanium dioxide is atitanium dioxide of rutile form or a mixture of the rutile and anataseforms.
 6. The body fluid purifying arrangement according to claim 1,wherein said metal or said oxide of said metal is in the form ofgranules or particles having a total porosity of at least about 50%. 7.The body fluid purifying arrangement according to claim 1, wherein saidmetal or said oxide of said metal is in the form of granules orparticles having a total surface area of at least about 0.01 m²/g. 8.The body fluid purifying arrangement according to claim 1, wherein saidmetal or said oxide of said metal is in the form of particles having anaverage particle diameter below 100 μm.
 9. The body fluid purifyingarrangement according to claim 1, wherein body fluid is selected from agroup consisting of blood, blood plasma, lymph fluid, cerebrospinalfluid, ascites and synovial fluid.
 10. A method of reducing 10 kDainterferon-γ inducible protein, IP-10, in a body fluid, said methodcomprising: pumping said body fluid past a first filter arranged inconnection with a fluid input of a purifying chamber comprised in a bodyfluid purifying arrangement according to claim 1 and into said purifyingchamber to contact said body fluid with granules or particles made of ametal or an oxide of a metal retained within said purifying chamber bysaid first filter and a second filter arranged in connection with afluid output of said purifying chamber to purify said body fluid fromIP-10; and pumping said body fluid purified from IP-10 out from saidpurifying chamber and past said second filter, wherein said metal is ametal of group 4 or 5 of the periodic table of the elements and selectedfrom the group consisting of titanium, zirconium, hafnium, niobium andtantalum.
 11. A body fluid purifying arrangement in the form of asyringe comprising: a purifying chamber having a common fluid input andfluid output; a filter arranged in connection with said common fluidinput and output; and granules or particles made of a metal or an oxideof a metal retained within said purifying chamber by said filter topurify a body fluid passing through said purifying chamber from 10 kDainterferon-γ inducible protein, IP-10, wherein said metal is a metal ofgroup 4 or 5 of the periodic table of the elements and selected from thegroup consisting of titanium, zirconium, hafnium, niobium and tantalum.12. The body fluid purifying arrangement according to claim 11, whereinsaid metal is selected from a group of titanium and tantalum.
 13. Thebody fluid purifying arrangement according to claim 11, wherein saidoxide of said metal is selected from a group consisting of an oxide oftitanium and an oxide of tantalum.
 14. The body fluid purifyingarrangement according to claim 13, wherein said oxide of titanium is atitanium dioxide.
 15. The body fluid purifying arrangement according toclaim 14, wherein said titanium dioxide is a titanium dioxide of rutileform or a mixture of the rutile and anatase forms.
 16. The body fluidpurifying arrangement according to claim 11, wherein said metal or saidoxide of said metal is in the form of granules or particles having atotal porosity of at least about 50%.
 17. The body fluid purifyingarrangement according to claim 11, wherein said metal or said oxide ofsaid metal is in the form of granules or particles having a totalsurface area of at least about 0.01 m²/g.
 18. The body fluid purifyingarrangement according to claim 11, wherein said metal or said oxide ofsaid metal is in the form of particles having an average particlediameter below 100 μm.
 19. The body fluid purifying arrangementaccording to claim 11, wherein body fluid is selected from a groupconsisting of blood, blood plasma, lymph fluid, cerebrospinal fluid,ascites and synovial fluid.
 20. A method of reducing 10 kDa interferon-γinducible protein, IP-10, in a body fluid, said method comprising:drawing a body fluid past a filter arranged in connection with a commonfluid input and output of a purifying chamber comprised in a body fluidpurifying arrangement according to claim 11 and into said purifyingchamber to contact said body fluid with granules or particles made of ametal or an oxide of a metal retained within said purifying chamber bysaid filter to purify said body fluid from IP-10; and pushing said bodyfluid purified from IP-10 out form said purifying chamber and past saidfilter wherein said metal is a metal of group 4 or 5 of the periodictable of the elements and selected from the group consisting oftitanium, zirconium, hafnium, niobium and tantalum.